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					Tumorigenesis, Infection and
       Calcification
    Or, things that can go wrong with implants


               Lecture 21
              April 7, 2009
                           Outline
• Tumorigenesis (section 4.7)
   – Review of concepts from previous lecture
   – Correlation between tumors with biomaterials
• Infection (section 4.8)
   – Review of concepts from previous lecture
   – Infections on or near biomaterials
   – Sterilization of biomaterials before implantation
• Calcification (section 6.4)
   – Pathologic calcification on biomaterial implants
       • Examples of common applications
       • Factors affecting calcification
       • Ways to prevent calcification
                           Part 1:
                        Tumorigenesis
• Neoplasia means “new growth” and is the process of excessive,
  unregulated and uncontrolled cell growth
• Neoplasma or tumor is the new growth
• Benign tumors
    – Do not penetrate adjacent tissues (organs) – self-limiting
    – Do not spread to distant sites
• Malignant tumors (cancers)
    – Invade contiguous tissue
    – Have the ability to enter the blood and lymph vessels
        • Therefore can spread to distant sites – Metastasis
• Malignant cells are dedifferentiated forms of the cells from which
  they originate
    – Aggressiveness increases with dedifferentiation
           Neoplastic Growth
• Neoplastic growth
  – Unregulated, therefore
  – Unrelated to the physiologic requirements of normal
    tissue, and
  – Unaffected by removal of the stimulus which caused it
• Neoplasms differ from:
  – Normal proliferation during fetal or postnatal growth
  – Normal wound healing following injury
  – Hyperplastic growth to adapt to a physiologic need,
    but that stops upon removal of the stimulus
    Basic Components of Tumors
• All tumors (benign and malignant) have two basic
  components:
   – Proliferating neoplastic cells that constitute their
     parenchyma (the essential parts of an organ or
     tissue that are concerned with function)
   – Supportive stroma made up of connective tissue
     and blood vessels
Tumorigenesis and Biomaterials
• Continuing debate over whether implants induce
  neoplasia (excessive and uncontrolled cell growth)
   – Studies with total hip replacements and breast
     implants show NO detectable increases in tumors
     at the implant site (Berkel et al., 1992; Mathieson,
     1995)
   – Su et al. Showed protective effect of silicone
     against breast cancer (1995)
   – Deapen and Brody showed increase in lung and
     vulvar cancer in patients with breast implants
     (1991)
   Implant-induced Neoplasms
• Predominantly sarcomas (cancers of connective
  tissue)
  – Characterized by rapid, locally infiltrative growth
  – Reported cases
     • Osteosarcoma near THA with UHMWPE or Ti-Co alloy (10
       yr, 3 yr implants, respectively)
     • Angiosarcoma near abdominal aortic graft with Dacron (12 yr
       implant)
     • Synovial sarcoma near THA with SS, PMMA (12 yr implant)
• Rarely carcinomas (cancers of epithelium) or
  lymphomas (cancers of blood cells)
     • Lymphoma with metal fracture fixation rod (17-yr implant)
        Pathology of Foreign Body
             Tumorigenesis
• The pathogenesis (origin) of implant-induced tumors is
  not well understood
   – Experimental data → physical effects not chemical
     characteristics determine tumorigenicity
• Tumors have been induced experimentally by many
  materials, including some „nonreactive‟ materials (such
  as glass, gold, and platinum)
   – High surface-area devices appear to be the most tumorigenic
   – Discontinuous surfaces (pulverized, shredded, woven,
     perforated) of biomaterials result in loss of tumorigenicity
• Solid-state tumorigenesis depends on the development
  of a fibrous capsule around the implant
   – A large inflammatory response inhibits tumor formation
             Hypothesized Step in Implant
                   Tumorigenesis
1. Cellular foreign-body reaction
2. Fibrous capsule formation
3. Preneoplastic cells contact implant surface during
   quiescent tissue reaction
    –   Dormancy and inactivity of attached macrophages
4. Preneoplastic cell maturation and proliferation
5. Tumor growth

•   Does not occur during active inflammation
•   Current hypothesis
    –   Foreign body does not initiate the tumor
         •   Preneoplastic cells develop prior to contact with implant
    –   However, contact with the foreign body is required for
        subsequent maturation and proliferation
            Foreign Body-Solid State
                 Carcinogenesis
• Possible mechanisms
    –   Less access to microvasculature
    –   Poorer diffusional supply of materials
    –   Reduced cell contact inhibition
    –   Difference in chemical and electrical environment
• Probability of tumor induction increases with increasing size of solid
• Probability of tumor induction varies inversely with the degree of
  inflammation response
    – Well tolerated implants have increasing incidence of carcinogenesis
    – Could the inflammation/immune response act to reduce the influence of
      changes
    – Could the presence of a fibrous capsule protect the surrounding tissue
      from carcinogenic activity
      Chemical Carcinogenesis
• Chemical carcingoenesis can occur as a result of 3 basic
  forms of stimulus
   – Near implants: direct presence of chemical in solution and
     diffusion to local tissues
   – Distant to implants: due to transport and concentration of
     chemicals produced by the implant
   – In the absence of an implant: due to inhalation, ingestion or
     absorption of chemicals
• Transformation mechanisms could be:
   – An alteration in the metabolic process of the cells
   – An alteration in the replication process of the cells
       • Growth stimulation
       • Reduction in contact inhibition
   – Mutagenesis
    Carcinogenic Biomaterials
• Polymers
  – Suspected to be due to chemical carcinogenesis
     • Pro-carcinogens
           – Polyvinylchloride (PVC) – produces vinyl halide
           – Polyvinylacetate – produces acetate
     • Complete carcinogens
           – Epoxide – possibly produced by many polymeric implants
• Metals
  – Act through either chemical (due to corrosion) or
    foreign body carcinogenesis
     • Studies support carcinogenic activity from
           –   Chromium
           –   Cobalt
           –   Nickel
           –   Iron
           –   Titanium
      Critical Factors in Neoplasm
                 Induction
• Implant configuration
   – Large surface area
• Fibrous capsules development
• A period of latency long enough to allow progression to
  neoplasia in a susceptible host
                      Part 2:
                     Infection
• Infection generally results from contamination
  during surgical procedures
• Implants must be effectively sterilized before use
• Device-related infections differ from acute
  bacterial infections in several ways
• Bacterial is naturally present on the surface of
  skin and in the body
  – The presence of bacteria does not necessarily
    indicate infection
                     Biofilms
• Examination of many biomaterials (from contact
  lenses to intrauterine devices to dental devices)
  showed biofilms on all
• Colonization vs infection
  – Colonization – presence of microbial biofilm
  – Infection – pathogenic response to biofilm
• In a study of 81 transcutaneous catheters
  – All had surfaces which were colonized by biofilms
  – Only 4 patients experienced overt infections
        Colonization of Bacteria




• Biofilms protect bacteria
   – Physical barrier to phagocytes
   – Inhibit T & B cell formation, antibody production (adaptive
     response)
   – Inhibit opsonization
   – Antibiotic ineffective
                            Infections
• Three types of infections
   – Superficial immediate – morganisms/bacteria on skin
   – Deep immediate – infection at implant soon after surgery,
     morganisms/bacteria on skin transported during implantation
   – Late infections – from months to years after surgery, not well understood,
     could be seeding of blood borne pathogens from another site
• Steps leading to infection
   – Bacterial attachment – reversible, nonspecific interactions with surface
     (~minutes)
   – Adhesion – irreversibly attached, nonspecific and specific receptor-ligand
     interactions (~hours)
   – Aggregation – bacteria divide and colonize, exude extracellular
     polysaccharide for protection (~ 1 day)
   – Dispersion – portions of colony are dispersed (shear flow, motion of
     implant, trauma, programmed release) to other areas of body, 2nd-ary
     infections
Bacterial Adhesion
 Bacterial Adhesion to Surfaces
• “Wild” bacteria are needed to understand
  adhesion to biomaterials
  – Bacteria adhere well to
     • Hydrophilic and hydrophobic surfaces
     • Smooth and rough surfaces
     • Smooth surfaces in high shear flows
  – No “perfect” material exists that resists bacterial
    colonization by surface properties alone
• Cultured bacteria do not behave the same as
  “wild” bacteria
  – Can lead to inaccurate conclusions in lab studies
           Bacterial Phenotypes
• Two phenotypes of bacteria differ significantly
   – Planktonic cells – individual, free cells
       • Are affected by antibiotics (which generally are selected because
         they act on this phenotype)
   – Biofilm cells – cells colonized on a surface
       • Are unaffected by antibiotics
       • Produce more exopolysaccharide
           – Becomes matrix (~85% of the colony volume)
           – Permanently attaches bacteria

• Biofilm colonies stabilize in 1-2 weeks and remain stable
  for years
   – Programmed detachment of planktonic cells induces local
     immune response
          Natural Control of Biofilm
                 Formation
• Control signaling mechanism
   – Signals sent between bacteria that lead to biofilms
• Prevention of phenotype change
   – Lock cells in planktonic phenotype which cannot attach to
     surfaces
• Marine plants are able to prevent biofilm formation
   – Possibly through a signal inhibitor
   – Ideal biomaterial would have similar abilities in vivo
• Engineering approaches
   – Ultrasound energy and weak DC fields leave biofilm colonies
     susceptible to conventional antibiotics
   – Modification of materials and designs
 Controlling Biofilm Colonization
• Agents
   – Antibiotics
       • Kill planktonic cells before they adhere
       • Can lead to resistant strains
   – Signal blockers to prevent attachment
       • Cells remain in the planktonic phenotype and are destroyed by
         immune system or antibiotics
• Delivery methods for above agents
   – Systemic therapy
   – Release of agents near device
   – Irrigation & other techniques that deliver agents to surface after
     implantation
Implant Geometry and Infection
• One design aspect that can affect infection
  rate is the geometry of the implant
  – Acellular spaces in implants
     • Acellular spaces offer little resistance to bacterial
       growth
     • Exposure to tissue and vascular processes is critical to
       fighting an infection
  – Possible acellular spaces in implants
     • Fibrous capsule
     • Dead spaces filled with acellular fluid
     • Porous structures
        – May provide dead space prior to ingrowth of tissue
      Features of Implant Associated
                 Infection
• Implanted biomaterial
• Adhesive bacterial colonization of the surface
• Resistance to host defense mechanisms and antibiotic
  therapy
• Characteristic bacteria such as S. epidermidis
• Specificity of phenomena (material, organism, host location)
• Transformation of nonpathogens into virulent organisms by
  presence of biomaterial
• Persistence of infection until biomaterial removal
• Absence of tissue integration
• Presence of tissue damage or necrosis
                     Sterility
• Absence of life as demonstrated by growth and
  reproduction
• Importance cannot be overestimated
   – Exposure to small quantities of bacterial can be
     serious
• Sterilization results in destruction of microorganisms
• Resistance of micro-organisms to sterilization is
   bacterial spores>fungal
     spores>fungi>bacteria>virus
• Sterility validation and assurance is one of the
  highest of the FDA‟s enforcement priorities
   Common Sterilization Methods
• Autoclaving - steam at 121 C + 15 PSI
  – Best understood and simple
  – Product must be moisture and heat stable
• Ethylene Oxide
  – Removal of ETO and byproducts must be
    demonstrated
  – TOXIC to workers and environment
• Gamma radiation
  – Easiest to quantify
  – Embrittles or discolors products
  – Expensive
                        Part 3:
                      Calcification
• Calcification or mineralization
   – Formation of calcium phosphate (CaP) or other
     calcium-containing compounds
   – Affects both synthetic and natural biomaterials
   – Natural process in the body (bone, teeth)
      • Can be desired for integration, osteoinductive materials
      • Can be pathologic (unintended, harmful, interferes with
        function) in many biomaterials or soft tissues
   – Intrinsic vs extrinsic calcification
      • Intrinsic occurs deep with in a tissue
      • Extrinsic occurs at the surface with attached cells and
        proteins
           Pathologic Calcification
• Can occur
   – In native tissue – e.g. atherosclerosis
   – Biomaterials – biological or artificial
• Mineral is apatite with a poor crystalline structure
   – Similar to calcium hydroxyapatite from bone
• Classification
   – Dystrophic – deposition of calcium salts in individuals with
     normal calcium metabolism
   – Metastatic – deposition of calcium salts in individuals with
     abnormal mineral metabolism
       • E.g. elevated blood Ca levels
• In young individuals, chemical environment of blood
  favors bone formation
   – Same environment can lead to calcification
          Common Examples (I)
• Heart Valves and Vascular Replacements
  – Gluteraldehyde pretreated porcine heart valves
     • Most significant dysfunction due to biomaterial calcification
     • Intrinsic calcification of the valve cusps causes tears,
       stiffening
     • More than 50% of porcine valves
       fail within 10-12 yrs in adults
     • Nearly all fail within 4 yrs in children
  – Allografts of valves or vascular
    segments
     • Calcification occurs at the wall
  – Synthetic vascular replacements
     • ePTFE & Dacron can calcify
              Common Examples (II)
• Soft contact lenses
   – Calcium deposits make lenses opaque
   – Difficult to remove without destroying the lens
• Polymeric bladder in blood pump
   – PU bladder surfaces may collect calcific deposits
       • Due to adherent proteins and cells
       • In surface defects from manufacturing or cracking
   – Leads to functional failure of the pump
• Breast implants
   – Calcification can occur in both silicone-gel implants and silicone
     envelopes filled with saline
   – Calcification increases with time
       • 11-20 yrs  26% of implants calcified
       • >23 yrs  nearly all implants calcified
    Assessing Biomaterial Calcification
       (I) Morphologic Techniques
• Allow for detection and characterization of microstructure,
  ultrastructure and distribution related to tissue/biomaterial
  structure
   – Macroscopic – gross examination
   – Radiograpy – assesses distribution of mineral
   – Microscopy
       • Low-power microscope
         shows distribution
       • Histology with calcium or
         phosphorus staining
         confirms location of mineral
         deposits, especially in initial
         stages
Assessing Biomaterial Calcification
(II) More Morphologic Techniques
– Electron microscopy
  • Transmission (TEM)
     – Observe the ultrastructure
       (sub-mm level) of
       calcification in thin section
     – Electron energy loss
       spectroscopy – localized
       elemental analysis
  • Scanning (SEM)
     – Images surface features
     – Energy dispersive x-ray
       analysis – semi-quantitative
       analysis of mineral
       deposition

                                       TEM images of calcification in (A)
                                       cells, (B) collagen.
 Assessing Biomaterial Calcification
     (III) Chemical Techniques
• Allow for characterization of progression & severity
  of deposition, and effectiveness of preventative
  treatments, but generally, sample configuration is
  destroyed
  – Atomic absorption spectroscopy – calcium quantification
  – Integrated coupled plasma (ICP) – hi-res quantification of
    Ca, Al, Fe ions in the same sample
  – Spectrophotometric detection – quantification of
    phosphate
  – X-ray diffraction – examination of crystal structure of CaP
             Pathophysiology (I)
• Regulation of calcification
   – Inhibition and procalcification are balanced differently depending
     on tissue
   – Many factors in blood and tissue affect calcification (normal and
     pathologic)
• Experimental models
   – In vitro testing – is possible, but not very useful
   – Subcutaneous model (small animal) – convenient, economical
     way to test host/material factors and mechanisms of
     mineralization
   – Valve implant model (large animal) – expensive, complex way
     to study promising materials/designs, elucidate failure
     processes, assess blood/surface interactions
             Pathophysiology (II)
• Bioprosthetic heart valve calcification
   – Mechanical factors seem to enhance, but not cause calcification
   – Neither inflammation nor immune response are likely the cause
     of calcification, though there is some correlation
   – Site of calcification generally dead cells or membrane fragments
     left from fixation (with gluteraldehyde)
   – Wall calcification (of aortic valve replacements) occurs heavily at
     the inner and outer surfaces (differs from mechanism in cusp)
• Collagen and elastin calcification
   – Calcification occurs to these structural proteins in various
     applications (tendon replacement, surgical sponges)
   – Stiffening limits usefulness
    Prevention of Calcification
• Techniques must
  – Not cause systemic or local toxicity
  – Not increase tendency toward thrombosis, infection,
    or structural degradation
  – Not affect normal performance (hemodynamics)
• Three approaches
  – Systemic therapy
  – Local therapy (drug delivery)
  – Biomaterial modification
  Advantages & Disadvantages of
      Prevention Techniques
• Systemic therapy
  – Advantage: has been shown to prevent calcification in animals
  – Disadvantage: affects normal calcification processes, stunts
    growth
• Local therapy (drug delivery)
  – Advantages: no systemic effects, but still limits calcification
  – Disadvantage: difficult to implement
• Biomaterial modification
  – Advantages: easy to implement (change to material not to host)
  – Disadvantages: not all approaches are well-studied, side-effects
    unknown
• Combination of approaches
                     Conclusions
• Tumorigenesis
   – Relationship between tumors is somewhat unknown
   – Device related tumors are rare, and causation generally cannot
     be demonstrated
• Infection
   – Normally harmless bacteria can create a biofilm on implanted
     materials, causing infections
   – Biofilms are difficult to remove once established – best to
     prevent formation
   – New materials/treatments use new knowledge of biofilms to
     prevent signaling, adhesion, etc
• Pathologic Calcification
   – Calcification of soft biomaterials can limit function
   – Once calcification begins, it is difficult to stop or reverse
   – Better understanding of mechanisms is improving ability to
     prevent calcification of natural and synthetic implants

				
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